EP1977001A1 - Oligonucleotide pour la detection de bacteries associees a la sepsie et jeux ordonnes de microechantillons pour la detection des bacteries mettant en oeuvre l'oligonucleotide - Google Patents

Oligonucleotide pour la detection de bacteries associees a la sepsie et jeux ordonnes de microechantillons pour la detection des bacteries mettant en oeuvre l'oligonucleotide

Info

Publication number
EP1977001A1
EP1977001A1 EP06715735A EP06715735A EP1977001A1 EP 1977001 A1 EP1977001 A1 EP 1977001A1 EP 06715735 A EP06715735 A EP 06715735A EP 06715735 A EP06715735 A EP 06715735A EP 1977001 A1 EP1977001 A1 EP 1977001A1
Authority
EP
European Patent Office
Prior art keywords
seq
sepsis
nos
bacteria
species
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP06715735A
Other languages
German (de)
English (en)
Other versions
EP1977001A4 (fr
Inventor
Cheol-Min Kim
Hee-Kyung Park
Hyun-Jung Jang
Eun-Sil Song
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
GENEIN Co Ltd
Original Assignee
GENEIN Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by GENEIN Co Ltd filed Critical GENEIN Co Ltd
Publication of EP1977001A1 publication Critical patent/EP1977001A1/fr
Publication of EP1977001A4 publication Critical patent/EP1977001A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria

Definitions

  • the present invention relates to oligonucleotides useful for detection and differential diagnosis of sepsis-causing bacteria and a detection method using the same, more particularly to a microarry comprising at least one of gram positive bacteria-specific and gram negative bacteria-specific oligonucleotides, sepsis-causing bacteria's genus-specific and species-specific oligonucleotides designed from the ITS target region which is hypervariable sequence of sepsis-causing bacteria as probes, and a detection method and a diagnosis kit using the same.
  • Sepsis is occurred by several microorganisms. Sepsis can be occured by enterence of microorganisms living together in stomach tube or tissue closed by skin, and partially infection of microorganisms in urogenital organ, bile, lung or stomach tube can induce blood infection. Microorganisms also can be directly infiltrated into blood througn an intravenous injection. In other words, a host human reaction appears variously by infection of such microorganisms, it is noticed as inflammation response such as high fever, slight fever, rigor, tachycardia and tachypnea.
  • the sepsis is a very fatal disease that progresses as severe sepsis, septic shock or MODS(multiple organ dysfunction syndrome) which brings about dysfunction of lung, kidney, liver and circulatory organ as a complication and makes a patient reach to the death when it can not diagnose the reason accurately in an early period. Therefore, it is very important that causing microorganisms are accurately separated from blood or infection region to diagnose sepsis accurately.
  • Sepsis can appear in everyone infected by the microorganism, especially sepsis appears as high frequency in inpatients such as a newborn baby, an old person, the people having lack of immunity, the people having a burn, the people having a wound by a traffic accident, an alcohol addict, drug addict and the people who takes a treatment using catheter in hospital.
  • Sepsis is a cause that at least the 18 milion persons progress to a severe sepsis every year, as sepsis is a typical incurable disease with 20 ⁇ 50% of mortality rate, and sepsis is a main reason of 14 hundred persons' death daily, it is reported that the frequency is 5-10 times than rectal or beast cancer. More than 215 thousends persons in USA and more than 135 thousends persons in Europe have died by diseases associated with sepsis every year. Sepsis is in 10 kinds of death reason containing cancer in USA. Such seriousness is caused by aging society, life extension of a chronic patient and the problem of AIDS and so on, it is predicted to be increased continuously in Korea.
  • DNA chip is in the spotlight as very useful technology, which can analyze many genes at once as they can attach a little genetic material on solid scaffold by using principle of pobe hybridization.
  • This identification method using molecular biological detecting technology is a technically very progressive method which can rapidly, sensitively and exactly detect at once in a cultured and clinical sample from bacteria with very slow proliferation rate and hardly culturing condition.
  • BMS in Korea provides the method which can rapidly and conveniently detect pathogenic bacteria than the method which requires a lot of times and high level technology. But this method is a limit to detect various bacteria using 16S rDNA gene of very complemental sequence as target region to distinguish species.
  • the application NO. 10-2002-0016679 can detect pathogenic bacteria by using multiplex PCR.
  • this can detect bacteria species less than bacteria species of the present invention, a main goal is the detection of pathogen which raises food poisoning and this is a drawback that has an effect on result of detection or has a contamination by a large number of primers in the method.
  • the Kor. Patent No. 10-2003-0005341 shows DNA chip technology detecting bateria associate with bacterial disease, this can detect 12 species of bacteria but there is no identical species to the present invention associated with sepsis.
  • the Kor. Patent No. 10-2005-7018087 as another patent, relates to nucleic acid probes for detecting pathogenic bacteria more than 44 species using DNA chip technology such as the present invention. But there is no identical species to ITS target region of the present invention associated with sepsis. There is using not ITS target region but 23 rDNA gene of base sequence of little diversity in some species able to detect identically to the present invention.
  • the present invention provides oligonucleotides for detecting gram posive and gram negative sepsis-causing bacteria, originated from ITS having hypervariable region which is base sequences of many variation.
  • another object of the present invention is to provide a microarray comprising oligonucleotides as probes for detection of sepsis-causing bacteria.
  • Another object of the present invention is to provide a method for identification and detection of sepsis-causing bacteria by using the miroarray.
  • Another object of the present invention is to provide a kit for identification and detection of sepsis-causing bacteria by using the microarray.
  • the present invention provides an oligonucleotide for gram positive-specific detection of sepsis-causing bacteria, comprising any one of one base sequence selected from SEQ ID Nos. 1 to 2 or its complementary sequence.
  • the present invention provides an oligonucleotide for gram negative-specific detection of sepsis- causing bacteria, comprising any one of one base sequence selected from SEQ ID Nos. 3 to 6 or its complementary sequence.
  • the present invention provides an oligonucleotide for genus-specific detection of sepsis-causing bacteria, comprising any one of one base sequence selected from SEQ ID Nos. 7 to 30 or its complementary sequence.
  • the present invention provides an oligonucleotide for species-specific detection of sepsis-causing bacteria, comprising any one of one base sequence selected from SEQ ID Nos. 31 to 104 or its complementary sequence.
  • the oligonucleotides of the present invention are designed based on multiple sequence alignment of ITS (internal transcribed spacer) hypervariable sequences of bacteria.
  • the oligonucleotides have specificity to the gram positive and gram negative bacteria, and can be used as primers for PCR amplification or probes for hybridization in order to specifically detect genus and species of the bacteria.
  • the present invention provides a gram positive-specific and gram negative-specific probe set and a genus-specific and species-specific probe set for detection of sepsis-causing bacteria, comprising more than one oligonucleotide selected from the above oligonucleotides.
  • the present invention provides a kit for diagnosing gram positive-specific and gram negative- specific sepsis-causing bacteria and genus-specific and species-specific sepsis- causing bacteria, comprising more than one oligonucleotide selected from the above oligonucleotides.
  • the oligonucleotides may be labeled with radioactive or non-radioactive labeling agent, the latter comprises conventional biotin,
  • the oligonucleotides can be used as primers or probes and the kit can comprise another primers for PCR amplification of the target DNA.
  • the present invention provides a microarray comprising more than one oligonucleotide selected from oligonucleotides for gram positive-specific and gram negative-specific detection and genus-specific and species-specific detection of the sepsis-causing bacteria, as the probes attached on a support.
  • the probes may be any materials having base sequence of the above oligonucleotides, preferably any one selected from a group consisting of DNA (Deoxyribose Nucleic Acid), RNA (Ribosse Nucleic Acid), and nucleic acid analogues selected from PNA (Peptide Nucleic Acid), LNA (Locked Nucleic Acid) and HNA (Hexitol Nucleic Acid).
  • the nucleic acid analogues is stable to enzymes such as nuclease, has structurally specific interaction with base sequence, and has advantage of stability in heat.
  • the probes can be manufactured for sense or antisense of the oligonucleotides. Therefore, the oligonucleotides have base sequence of the SEQ ID Nos. or its complementary sequence.
  • the probes can further comprise bacteria specifc nucleotides which is commonly present in bacteria and well-known in the art.
  • the probes can further comprise a bacteria specific oligonucleotide such as SEQ ID No. 46 of Kor. Patent No. 2004-68313, which is commonly present in 23S rDNA base sequence of the bacteria.
  • the support may be made of any one selected from a group consisting of slide glass, plastic, membrane, semiconductive chip, silicon, gel, nano material, seramic, metal material and optical fiber or those mixture.
  • the microarray of the present invention can be manufactured using conventional method such as a pin microarray (Microarray printing technology, Don Rose, Ph.D., Cartesian Technologies, Inc., Anal Biochem, 320(2):281-91(2003)), a ink jet (Nat Biotech, 18;438-441(2000), Bioconjug Chem,13(1 );97-103(2002)), photolithography (Cur Opinion Chem Biol, 2;404-410(1998), Nature genetics supplement, 21 :20-24(1999)) or a electric array method (Ann Biomed Eng.
  • the microarray of the present invention may further comprise an extraction agent for isolating target DNA, a PCR kit containing primers for amplifying target gene, a hybridization reaction buffer, a washing solution for the unhybridized DNA, a cover slip, dyes, a washing solution for unbound dyes and a description sheet for the microarray, except for the microarray of the present invention.
  • the present invention provides a method for detection, comprising the following steps: a) purifying nucleic acids from a sample; b) amplifying target DNA among the purified nucleic acid; c) hybridizing the amplified target DNA with probes of the microarray according to the present invention; and d) detecting signals generated from the formed hybrid.
  • the step b) for amplifying target DNA can be performed using Hot-start PCR, Nested PCR, Multiplex PCR, RT- PCR (reverse transcripase PCR), DOP (degenerate oligonucleotide primer) PCR, Quantitive RT-PCR, In-Situ PCR, Micro PCR, modified PCR such as Lab-on a chip PCR and isothermal amplification method such as RCA (rolling circle amplification) as well as general PCR reaction.
  • the detection method of the present invention can be performed using probe amplification or signal amplification reaction such as tyramide signal amplification (Nucleic Acids Res. 30;e4(2002)), nanoparticle probe, Raman-active dye (Science, 297; 1536-1540(2002)) and branched DNA.
  • the sample may be bloods, body fluids, tissues, sputum, excreta, urine or discharge.
  • the purifying step a) can be performed using conventional DNA or RNA purification method or kit.
  • the amplifying step b) can be performed using conventional PCR method.
  • the detection of the PCR product can be performed using conventional electrophoresis wih agarose gel.
  • the signal detecting step d) can be performed using conventional fluorescence scanner after binding with conventional dyes such as Cy5 or Cy3.
  • a method for simultaneously genotying and detecting more than one sepsis-causing bacteria species selected from a group consisting of the following members: a) gram positive bacteria (SEQ ID Nos. 1 to 2) and gram negative bacteria (SEQ ID Nos. 3 to 6); b) Bacteroides genus (SEQ ID Nos. 7 to 10) and Bacteriodes species (SEQ ID Nos. 31 to 40); c) Enterococcus genus (SEQ ID Nos. 11 to 12) and Enterococcus species
  • the present invention provides a method for gram positive-specific and gram negative-specific detection of bateria associated with sepsis and a method for detecting one or more bacteria species simultaneously.
  • the present invention provides a method for detection using SBE (Single base extension), Sequencing, RFLP (Restriction fragment length polymorphism), or REA (Restriction endonuclease analysis) based on difference of one base sequence by using the oligonucleotides which is designed for gram positive-specific and gram negative- specific detection of sepsis-causing bacteria and for genus-specific and species- specific detection of sepsis-causing bacteria.
  • SBE Single base extension
  • Sequencing Sequencing
  • RFLP Restriction fragment length polymorphism
  • REA Restriction endonuclease analysis
  • the method for detecting existence of sepsis-causing bacteria and identifying gram positive-specific and gram negative bacteria and genus-specific and species- specific bacteria using the microarray of the present invention comprises the following steps: a) if necessary, purifying nucleic acids from a cultured or clinical sample;
  • the probes have a diversity of probe composition and more than one probes.
  • the probes are optimized to simultaneously hybrid with its target region under the same hybrid and washing condition which can detect gram positive and gram negative bacteria and genus and species of the sepsis-causing bacteria at once.
  • the present invention provides a microarray comprising probes set for detecting gram positive-specific and gram negative-specific bacteria and genus and species of the sepsis-causing bacteria attached on the support, which can simultaneously detect gram positive and gram negative bacteria and identify genus and species of the bacteria as quickly and exactly as passible from only one experiment of a sample.
  • FIG. 1 shows a schematic flow chart of the prevent invention.
  • FIG. 2 shows location of tartet region, primers and probes used for amplifying sepsis-causing bacteria from biological sample.
  • FIG. 3 show a microarray comprising a probe set consisting of bacteria universal probes and genus-specific and species-specific probes for detecting bactera associated with sepsis, attached on a support.
  • FIG. 4 shows results of hybridization reaction of bacterial common probes and genus-specific and species-specific probes for Streptococcus bovis among sepsis- causing bacteria.
  • FIG. 5 shows results of hybridization reaction of bacterial common probes and genus-specific and species-specific probes for Staphylococcus saprophyticus among sepsis-causing bacteria.
  • FIG. 6 shows results of hybridization reaction of bacterial common probes and genus-specific and species-specific probes for Enterococcus faecium among sepsis- causing bacteria.
  • FIG. 7 shows results of hybridization reaction of bacterial common probes and genus-specific and species-specific probes for Listeria monocytogenes among sepsis- causing bacteria.
  • FIG. 8 shows results of hybridization reaction of bacterial common probes and genus-specific and species-specific probes for Escherichia coli among sepsis-causing bacteria.
  • FIG. 9 shows results of hybridization reaction of bacterial common probes and genus-specific and species-specific probes for Enterobacter aerogenes among sepsis- causing bacteria.
  • FIG. 10 shows results of hybridization reaction of bacterial common probes and genus-specific and species-specific probes for Klebsiella pneumoniae among sepsis- causing bacteria.
  • FIG. 11 shows results of hybridization reaction of bacterial common probes and genus-specific and species-specific probes for Serratia marcescens among sepsis- causing bacteria.
  • FIG. 12 shows results of hybridization reaction of bacterial common probes and genus-specific and species-specific probes for Pseudomonas aeruginosa among sepsis-causing bacteria.
  • FIG. 13 shows results of hybridization reaction of bacterial common probes and genus-specific and species-specific probes for Bacteroides thetaiotaomicron among sepsis-causing bacteria.
  • FIG. 14 shows results of hybridization reaction of bacterial common probes and genus-specific and species-specific probes for Streptococcus intermedius among sepsis-causing bacteria.
  • FIG. 15 shows results of hybridization reaction of bacterial common probes for Salmonella bongori unrelated with sepsis.
  • FIG. 16 shows results of hybridization reaction of bacterial common probes for Salmonella bongori unrelated with sepsis.
  • FIG. 17 shows a microarray comprising a probe set consisting of bacteria universal probes, gram-positive and gram-negative probes and genus-specific and species-specific probes attached on a support for more specific detection of the sepsis-causing bacteria.
  • FIG. 18 shows results of hybridization reaction of bacterial common probes, gram positive-specific probes and genus-specific and speies-specific probes for Streptococcus pneumoniae pertaining to gram-positive sepsis-causing bacteria.
  • FIG. 19 shows results of hybridization reaction of bacterial common probes and gram positive-specific probes for Bacillus cereus of gram-positive bacteria unrelated with sepsis.
  • FIG. 20 shows results of hybridization reaction of bacterial common probes, gram negative-specific probes and genus-specific and speies-specific probes for Serratia marcescens pertaining to gram-negative sepsis-causing bacteria.
  • FIG. 21 shows results of hybridization reaction of bacterial common probes, gram negative-specific probes and genus-specific and speies-specific probes for Pseudomonas stuzen pertaining to gram-negative sepsis-causing bacteria.
  • FIG. 1 shows a schematic flow chart of the prevent invention.
  • the method according to the present invention comprises designing specific probes for detecting gram positive and gram negative sepsis-causing bacteria, extracting DNA from cultured and clinical sample, amplifying nucleic acid by PCR, and detecting accurately and simultaneously gram-positive and gram-negative bacteria and genus and species of bacteria with existence of sepsis-causing bacteria by using the microarray.
  • FIG. 2 shows location of the target region for amplifying bacteria in biological sample, primers for amplifying and probes.
  • FIG. 2 shows, in gene structure of bacteria, ITS of base sequence hypervariable region, location of primers belong to bacteria which can amplify ITS, specific probes designed by present inventor for gram positive and gram negative bacteria and specific location of probes for genus and species.
  • New oligonucleotides for gram positive-specific probes of sepsis-causing bacteria developed in the present invention are as shown in Table 1. [Table 1] New probes for gram positive-specific detection of sepsis-causing bacteria
  • New oligonucleotides for gram negative-specific probes of sepsis-causing bacteria developed in the present invention are as shown in Table 2. [Table 2] New probes for gram negative-specific detection of sepsis-causing bacteria
  • New oligonucleotides for probes detecting genus-specific sepsis-causing bacteria developed in the present invention are as shown in Table 3. [Table 3] New probes for bacteria genus-specific detection of sepsis-causing bacteria
  • New oligonucleotides for probes detecting species-specific sepsis-causing bacteria developed in the present invention are as shown in Table 4. [Table 4] New probes for bacteria species-specific detection of sepsis-causing bacteria
  • Example 1 Incubation of Bacteria and isolation of Genomic DNA Total 56 kinds of strains were obtained from the American Type Culture
  • ATCC American Type Culture Collection
  • the strains were selected in each culturing media under each culturing conditions according to manual provided by ATCC. From the cultured media, strain colonies were obtained with a white gold ear and input 1.ml tube, 10OuI of InstaGene matrix (Bio-Rad, USA) was added thereto and suspended, and reaction was performed at 56 0 C for 30 minutes in constant temperature bath. And then, the reactant was shook for 10 seconds, heated at 100 0 C for 8 min, shook again for 10 sec, centrifuged at 12,000 rpm for 3 min, recovered DNA. The strains used were as followed Tabel 5. : [Table 5]
  • probes used for detection of sepsis-causing bacteria is confirmed specificity of probes by multiple alignment and BLAST searching as selecting ITS target base sequence of sepsis-causing bacteria published in Genbank.
  • gram positive-specific probes of sepsis-causing bacteria is only complement ary in species of gram-positive bacteria, is selected from base sequence having very lower similarity to other species.
  • gram negative-specific probes is only complementary in species of selected gram-negative bacteria, is selected base sequence having very lower similarity to other species.
  • genus-specific probes of sepsis-causing bacteria is only complementary in specis of each genus, is selected from base sequence having very lower similarity to specis of other genus.
  • species-specific probes is only complementary in each species, is selected from base sequence having very lower similarity to species of other species.
  • Designed gram-positive bacteria, gram-negative bacteria and genus and specis-specific probes were standed for in Table 1 to Table 4. Above all probes can be used as not being limited in base sequence of Tabel 1 to Table 4 but being designed primers and probes consisted of base sequence comprising it.
  • Example 3 Preparation of taget DNA
  • Reaction composition is added to water to be 25ul of total volume after adding 10D PCR buffer(100 mM KCI 1 20 mM Tris HCI (pH 9.0), 15 mM MgCI 2 ) 5 ⁇ l, dNTP(deoxynucleoside triphosphates) mixture (dATP, dGTP, dTTP, and dCTP each 10 mM) 1 ⁇ l, forward and backward primers (each 10 pmole) each 1 ⁇ l, Taq polymerase (5 units// ⁇ , QIAGEN, Inc., Valencia, USA) 0.2 ⁇ l, template DNA 4 ⁇ l.
  • Reaction condition is denaturation at 94 0 C for 3 minutes, denaturation at 94 0 C for 1 minute. Annealing reaction is carried out at 50 ° C for 1 minute, extension reaction is carried out at 72 0 C for 1 minute, and we repeated 30 times these process.
  • Example 4 Probe immobilization on support
  • the probes prepared in Example 2 is hybrided by 9 Guanine bases, 3 Amine base of spacer and 15 ⁇ 25 base sequence in 5' end.
  • the common probes which can know existence of bacteria used in experiment is directly developed by the present inventor, is used base sequence (Uni-459 : 5'-CCG ATA GTG AAC CAG TAC C- 3'XPatent No. 04-68313, SEQ ID No. 46) being applying for a patent.
  • the slide fixes all probes on surface of support using BMT Guanine ChipTM(Biomatrix Technology Co., Ltd. Korea) recognizing guanine and fixing biological molecular on support, then the slide is manufactured by washing non-fixed probes.
  • Example 5 Hybridization
  • the biotin-labeled target products prepared in Example 3 were thermally treated to be denaturated in to single strands and cooled to 4 0 C.
  • reaction solution containing Cy5- streptavidin or Cy3- streptavidin(Amersham pharmacia biotech, USA) and 60ml of hybridization reaction solution comprising 1 ⁇ 5ul of target DNA.
  • This hybridization reaction solution was portioned on the slide glass after probes attachment and washing, and the slide glass was covered with a cover seal and reacted at 40 0 C for 30 minutes.
  • the hybridized result was scanned using a non-confocal laser scanner (GenePix 4000Am, Axon Instruments, USA) and analyzed by image analysis. The result is standed for follow Table 6.
  • FIG. 3 show a microarray comprising a support, as a probe set, consisting of bacteria universal probes for detecting bactera associated with sepsis and genus- specific and species-specific probes.
  • FIG. 4 shows results of hybridization reaction for Streptococcus bovis using mircoarray, and shows scan image of bacteria common probes (Patent No. 04-68313 SEQ ID NO. 46) and Streptococcus genus-specific (SEQ ID No. 29) and Streptococcus vobis species-specific probes (SEQ ID No. 102).
  • FIG. 5 shows results of hybridization reaction for Staphylococcus saprophytics using mircoarray, and shows scan image of bacteria common probes (Patent No. 04- 68313 SEQ ID NO. 46) and Staphylococcus genus-specific (SEQ ID No. 27) and Staphylococcus saprophytics species-specific (SEQ ID No. 94) probes.
  • FIG. 6 shows results of hybridization reaction for Enterococcus faecium using mircoarray, and shows scan image of bacteria common probes (Patent No. 04-68313 SEQ ID NO. 46) and Enterococcus genus-specific (SEQ ID No. 11) and Enterococcus faecium species-specific (SEQ ID No. 43) probes.
  • FIG. 7 shows results of hybridization reaction for Listeria monocytogenes using mircoarray, and shows scan image of bacteria common probes (Patent No. 04-68313 SEQ ID NO. 46) and Listeria genus-specific (SEQ ID No. 18) and Listeria monocytogenes species-specific (SEQ ID No. 73) probes.
  • FIG. 8 shows results of hybridization reaction for Escherichia coli using mircoarray, and shows scan image of bacteria common probes (Patent No. 04-68313 SEQ ID NO. 46) and Escherichia genus-specific (SEQ ID No. 46) and Escherichia coli species-specific (SEQ ID No. 56) probes.
  • FIG. 9 shows results of hybridization reaction for Enterobacter aerogenes using mircoarray, and shows scan image of bacteria common probes (Patent No. 04-68313 SEQ ID NO. 46) and Enterobacter genus-specific (SEQ ID No. 17) and Enterobacter aerogenes species-specific (SEQ ID No. 50) probes.
  • FIG. 10 shows results of hybridization reaction for Klebsiella pneumoniae using mircoarray, and shows scan image of bacteria common probes (Patent No. 04-68313 SEQ ID NO. 46) and Klebsiella genus-specific (SEQ ID No. 17) and Klebsiella pneumoniae species-specific (SEQ ID No. 67) probes.
  • FIG. 11 shows results of hybridization reaction for Serratia marcescens using mircoarray, and shows scan image of bacteria common probes (Patent No. 04-68313 SEQ ID NO. 46) and Serratia genus-specific (SEQ ID No. 25) and Serratia marcescens species-specific (SEQ ID No. 87) probes.
  • FIG. 12 shows results of hybridization reaction for Pseudomonas aeruginosa using mircoarray, and shows scan image of bacteria common probes (Patent No. 04- 68313 SEQ ID NO. 46) and Pseudomonas genus-specific (SEQ ID No. 21) and Pseudomonas aeruginosa species-specific (SEQ ID No.
  • FIG. 13 shows results of hybridization reaction for Bacteroides thetaiotaomicron using mircoarray, and shows scan image of bacteria common probes (Patent No. 04- 68313 SEQ ID NO. 46) and Bacteroides genus-specific (SEQ ID No. 7) and Bacteroides thetaiotaomicron species-specific (SEQ ID No. 35) probes.
  • FIG. 14 shows, even though bacteria unrelated with sepsis, results of hybridization reaction for Streptococcus intermedius using mircoarray, and shows scan image of bacteria common probes (Patent No. 04-68313 SEQ ID NO. 46) and
  • FIG. 15 shows, even though pathogenic bacteria unrelated with sepsis, results of hybridization reaction for Salmonella bongori using mircoarray, and shows scan image reacting to bacteria common probes (Patent No. 04-68313 SEQ ID NO. 46) and not reacting to specific probes for detection of sepsis-causing bacteria.
  • FIG. 16 shows, even though pathogenic bacteria unrelated with sepsis, results of hybridization reaction for Acinetobacter baumannii using mircoarray, and shows scan image reacting to bacteria common probes (Patent No. 04-68313 SEQ ID NO. 46) and not reacting to specific probes for detection of sepsis-causing bacteria.
  • FIG. 17 shows detection for the existence of sepsis-causing bacteria, and shows microarray comprising a support, as a probe set, consisting of bacteria common probes, gram-positive and gram-negative probes and genus-specific and species- specific probes.
  • FIG. 18 shows results of hybridization reaction for Streptococcus pneumoniae of gram-positive bacteria among sepsis-causing bacteria using mircoarray, and shows scan image of bacteria common probes (Patent No. 04-68313 SEQ ID No. 46), gram- positive probes (SEQ ID No. 1-2) and Streptococcus genus-specific (SEQ ID No. 29) and Streptococcus pneumoniae species-specific (SEQ ID No. 98) probes.
  • FIG. 19 shows results of hybridization reaction for Bacillus cereus of gram- positive bacteria among bacteria unralated with sepsis using mircoarray, and shows scan image reacting to bacteria common probes (Patent No. 04-68313 SEQ ID NO. 46) and gram-positive specific probes (SEQ ID No. 1-2) and not reacting to specific probes for detection of sepsis-causing bacteria.
  • FIG. 20 shows results of hybridization reaction for Serratia marcescens of gram-negative bacteria among sepsis-causing bacteria using mircoarray, and shows scan image reacting to bacteria common probes (Patent No. 04-68313 SEQ ID NO.
  • FIG. 21 shows results of hybridization reaction for Pseudomonas stuzeri of gram-negative bacteria among sepsis-causing bacteria using mircoarray, and shows scan image reacting to bacteria common probes (Patent No. 04-68313 SEQ ID NO.
  • composition and layout of each probes can be changed because this is only tipical example of probes layout among new oligonucleotides designed in the present invention.
  • the present invention developed the miroarray and the diagnosis kit for detecting sepsis-causing bacteria comprising any one selected from a group consisting of gram positive- and gram negative-specific and genus- and species- specific probes designed from ITS of base sequence hypervariable region of the bacteria, and comfirmed its specificity.
  • the present invention can provide an antibiotics therapy for accurately removing infectious agent related to sepsis by detecting existence of sepsis-causing bacteria
  • the present invention can prevent a patient from abuse and misuse of antibiotics and decrease time of hospital treatment and medical cost of the patient. Further, the present invention has advantage

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne des oligonucléotides pour la détection de bactéries entraînant la sepsie et un procédé de détection mettant en oeuvre les oligonucléotides, plus particulièrement un jeu ordonné de microéchantillons comportant au moins un parmi des oligonucléotides spécifiques de bactéries à gram positif et spécifiques de bactéries à gram négatif, des oligonucléotides spécifiques de genres et des oligonucléotides spécifiques d'espèces de bactéries entraînant la sepsie conçus à partir de la région de l'espaceur interne transcrit (ITS) qui est une séquence de base hypervariable de bactéries entraînant la sepsie en tant que sondes, et un procédé de détection et une trousse de diagnostic mettant en oeuvre lesdits oligonucléotides. Selon la présente invention, une thérapie antibiotique peut être fournie par l'invention pour l'élimination précise d'agent infectieux associé à la sepsie par la détection de l'existence de bactéries entraînant la sepsie et l'identification de bactéries à gram positif et à gram négatif et de genres et d'espèces des bactéries simultanément. La présente invention permet la prévention d'abus et de mauvais usage par un patient d'antibiotiques et de réduire le temps de traitement hospitalier et les frais médicaux du patient. La présente invention présente en outre l'avantage d'empêcher des complications et de réduire le taux de mortalité.
EP06715735A 2006-01-20 2006-01-20 Oligonucleotide pour la detection de bacteries associees a la sepsie et jeux ordonnes de microechantillons pour la detection des bacteries mettant en oeuvre l'oligonucleotide Withdrawn EP1977001A4 (fr)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/KR2006/000237 WO2007083852A1 (fr) 2006-01-20 2006-01-20 Oligonucléotide pour la détection de bactéries associées à la sepsie et jeux ordonnés de microéchantillons pour la détection des bactéries mettant en oeuvre l'oligonucléotide

Publications (2)

Publication Number Publication Date
EP1977001A1 true EP1977001A1 (fr) 2008-10-08
EP1977001A4 EP1977001A4 (fr) 2009-12-02

Family

ID=38287790

Family Applications (1)

Application Number Title Priority Date Filing Date
EP06715735A Withdrawn EP1977001A4 (fr) 2006-01-20 2006-01-20 Oligonucleotide pour la detection de bacteries associees a la sepsie et jeux ordonnes de microechantillons pour la detection des bacteries mettant en oeuvre l'oligonucleotide

Country Status (3)

Country Link
US (1) US20090286691A1 (fr)
EP (1) EP1977001A4 (fr)
WO (1) WO2007083852A1 (fr)

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2694526A4 (fr) * 2011-04-01 2015-04-22 Advandx Inc Coloration moléculaire de gram
AU2012344703A1 (en) * 2011-12-01 2014-07-24 Geneohm Sciences Canada, Inc. Molecular assay for the amplification and detection of KPC genes responsible for high-level resistance to carbapenem in gram negative bacteria
US20150344973A1 (en) * 2012-04-23 2015-12-03 Pathogenica, Inc. Method and System for Detection of an Organism
US10260111B1 (en) * 2014-01-20 2019-04-16 Brett Eric Etchebarne Method of detecting sepsis-related microorganisms and detecting antibiotic-resistant sepsis-related microorganisms in a fluid sample
KR102204327B1 (ko) * 2017-06-30 2021-01-19 한국화학연구원 패혈증 진단용 키트 및 이를 이용한 진단방법
US20190062809A1 (en) 2017-08-24 2019-02-28 Clinical Micro Sensors, Inc. (dba GenMark Diagnostics, Inc.) Electrochemical detection of bacterial and/or fungal infections
WO2019040769A1 (fr) 2017-08-24 2019-02-28 Clinical Micro Sensors, Inc. (dba GenMark Diagnostics, Inc.) Détection électrochimique d'infections bactériennes et/ou fongiques
US11287422B2 (en) 2019-09-23 2022-03-29 Element Biosciences, Inc. Multivalent binding composition for nucleic acid analysis
WO2022029159A1 (fr) * 2020-08-04 2022-02-10 Theraxen S.A. Dosage d'adn ramifié à base de xéno-nucléotides pour la détection d'acides nucléiques
CN113293221B (zh) * 2021-02-01 2022-07-12 重庆市急救医疗中心(重庆市第四人民医院、重庆市急救医学研究所) 一种人类脓毒症病原体检测试剂盒及检测方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997032038A2 (fr) * 1996-02-27 1997-09-04 Oulutech Ltd. Diagnostic des mammites base sur des sequences specifiques d'adn dans le lait
WO2004053155A1 (fr) * 2002-12-06 2004-06-24 Roche Diagniostics Gmbh Detection par essais multiplex d'organismes pathogenes
WO2004052606A2 (fr) * 2002-12-06 2004-06-24 Innogenetics N.V. Detection, identification et differenciation de taxa eubacteries par dosage biologique d'hybridation

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20030087912A (ko) * 2002-05-09 2003-11-15 (주)메디제네스 Ｉｔｓ로부터 유도된 세균성 감염질환 원인균의 검출용ｄｎａ탐침

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997032038A2 (fr) * 1996-02-27 1997-09-04 Oulutech Ltd. Diagnostic des mammites base sur des sequences specifiques d'adn dans le lait
WO2004053155A1 (fr) * 2002-12-06 2004-06-24 Roche Diagniostics Gmbh Detection par essais multiplex d'organismes pathogenes
WO2004052606A2 (fr) * 2002-12-06 2004-06-24 Innogenetics N.V. Detection, identification et differenciation de taxa eubacteries par dosage biologique d'hybridation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of WO2007083852A1 *

Also Published As

Publication number Publication date
WO2007083852A1 (fr) 2007-07-26
US20090286691A1 (en) 2009-11-19
EP1977001A4 (fr) 2009-12-02

Similar Documents

Publication Publication Date Title
WO2007083852A1 (fr) Oligonucléotide pour la détection de bactéries associées à la sepsie et jeux ordonnés de microéchantillons pour la détection des bactéries mettant en oeuvre l'oligonucléotide
KR100850193B1 (ko) 모든 세균의 감별을 위한 세균 특이적, 속 특이적 및 종특이적 올리고뉴클레오티드, 이를 포함하는 진단 키트, 및이를 이용한 검출 방법
EP0452596A1 (fr) Sondes d'hybridation provenant de régions d'espacement entre les gènes de rRNA 16S et 23S pour la détection des micro-organismes non viraux
US20100273159A1 (en) Nested Multiplex Amplification Method for Identification of Multiple Biological Entities
JP7281504B2 (ja) 皮膚糸状菌症の診断のためのアッセイ
EP2078756A1 (fr) DÉTECTION DE BACTÉRIES PAR L'UTILISATION DU GÈNE DnaJ ET UTILISATION DU PROCÉDÉ
WO2009005178A1 (fr) Microréseau pour la détection et l'identification de microorganismes associés à des maladies périodontiques et procédé de diagnostic de maladies orales infectieuses à l'aide du microréseau
WO2008135931A2 (fr) Nécessaire et procédé de détection et d'identification de levures cliniquement importantes, à l'aide d'une amplification d'adn isothermique suivie par l'hybridation à des sondes oligonucléotidiques spécifiques d'une espèce, et applications respectives
EP2240605B1 (fr) Sondes d'acides nucléiques et amorces à large portée pour la détection des bactéries infectieuses
EP1969145B1 (fr) Micropuce oligonucleotidique et methode destinees a l'identification d'agents pathogenes
US20120171681A1 (en) Oligonucleotides, methods and kits for detecting and identifying vancomycin-resistant enterococcus
JP2002537824A (ja) 細菌の同定
JP5916740B2 (ja) 核酸標的の定量的多重同定
JP4744053B2 (ja) マイコバクテリウム属（Ｍｙｃｏｂａｃｔｅｒｉｕｍ）種の核酸増幅および検出
TW202144584A (zh) 克雷柏氏菌屬細菌之檢測用之引子組及探針
KR101623489B1 (ko) 성인성 질환의 원인균 탐지용 고감도 프로브 및 이를 포함하는 키트
JP2010515451A (ja) 大腸菌検出用ｄｎａチップ
WO2004046379A9 (fr) Sondes d'acide nucleique et amorces a large spectre provenant de regions de genes de la topoisomerase, et procedes utilisant celles-ci
JP2017189135A (ja) 乳酸菌検出用担体、及び乳酸菌の検出方法
US20090197775A1 (en) Nuclease on chip
ES2253279T3 (es) Metodos y composiciones para la deteccion de especies del complejo de mycobacterium avium.
WO1998010073A1 (fr) Procede et materiau de detection de champignons
KR20080104118A (ko) 패혈증 관련 질환 원인 세균의 감별 및 동정을 위한올리고뉴클레오티드, 이를 포함하는 마이크로어레이와 이를이용한 검출 방법
KR101338292B1 (ko) Ａ형 간염 바이러스에 대한 역전사 중합효소 연쇄반응 효소면역 측정법
KR101518561B1 (ko) 아시네토박터 균종 검출을 위한 종-특이 ｐｃｒ 기법 개발

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20080723

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI SK TR

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: PARK, HEE-KYUNG

Owner name: KIM, CHEOL-MIN

Owner name: GENEIN CO., LTD.

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: PARK, HEE-KYUNG

Owner name: GENEIN CO., LTD.

Owner name: KIM, CHEOL-MIN

A4 Supplementary search report drawn up and despatched

Effective date: 20091103

17Q First examination report despatched

Effective date: 20100305

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20100916